Osteoporosis is a skeletal disorder characterised by diminished bone mass, decreased bone mineral density (BMD), decreased bone strength and associated with an increased risk of bone fracture (Lane J. M., et al., Clin. Orthop. Relat. Res., 372, 2000, 139-150). Osteoporotic fractures most often occur in the vertebrae, hips or the femoral neck. These fractures severely impair the quality of life because of pain, long-lasting immobility and poor recovery. Bone comprises of several different cell types. Osteoblast (bone formation) lays down new bone from the mineral present in the extracellular milieu around the cells. Osteoclasts (bone loss) remove old bones, releasing the minerals compiled within bone back into the extracellular matrix. The balance between adequate new bone being deposited and old bone being removed is what gives bone its extremely beneficial properties. Osteoporosis occurs when the rate of the bone resorption is greater than the rate of bone formation (Seeman E., et al., N. Engl. J. Med., 354(21), 2006, 2250-2261). Postmenopausal estrogen deficiency is the most common cause of osteoporosis in women, as estrogen puts a break on osteoclast lifespan. Other major risk factors in the development of osteoporosis include: low calcium intake, vitamin D deficiency, type-1 diabetes, rheumatoid arthritis, long-term use of medication such as anticonvulsants and corticosteroids and low levels of testosterone in men (Cole Z. A., et al., Curr. Rheumatol. Rep., 10(2), 2008, 92-96; Harvey, N., et al., Cliff. Rheumatol. Rep., 5(1), 2003, 75-81).
Patients with osteoporosis would benefit from new therapies designed to promote fracture repair or from therapies designed to prevent or lessen the fractures associated with the disease (Lindsay R., Lancet, 341(8848), 1993, 801-805). At present, there is no effective cure for osteoporosis, though estrogen, raloxifene (oestrogen receptor modulators), calcitonin and the bisphosphonates (etidronate, alendronate and risedronate) are used to treat the disease with varying levels of success through their action to decrease bone resorption (Recker R. R., J. Clin. Endocrinol. Metab., 1993, 76(1), 14-16).
Native human Parathyroid Hormone (PTH) is an 84 amino acids polypeptide that acts as the most important regulator of calcium homeostasis in the human body through its direct action on bone and kidneys (Kronenberg H. M., Bringhurst F. R., Nussbaum S. R., Jüppner H., Abou-Samra A. B., In Handbook of Experimental Pharmacology, Mundy, G. R., and Martin, T. J., (eds), pp. 185-201, Springer-Verlag, Heidelberg (1993)). PTH synthesis and release from the parathyroid glands are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH in-turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood. PTH contributes to net gastrointestinal absorption of calcium by favouring the renal synthesis of the active form of vitamin D. PTH promotes calcium reabsorption from bone, indirectly by stimulating differentiation of the osteoclasts (bone-resorbing cells). Administration of PTH via parenteral route efficiently increases bone mineral density (BMD), bone strength and reduces the incidence of new osteoporotic fractures in osteoporotic patients (Greenspan S. L., et al., Ann. Intern. Med., 146(5), 2007, 326-339; Neer R. M., et al., N. Engl. J. Med., 344, 2001, 1434-1441).
PTH exerts all these effects primarily through its interaction with a cell surface PTH receptor, which is expressed in numerous tissues, most abundantly in kidney, bone and growth plate chondrocytes (Lanske B., et al., Crit. Rev. Eukaryot. Gene Expr., 8, 1998, 297-320). The PTH receptor is homologous in primary structure to a number of other receptors that bind peptide hormones, such as secretin, calcitonin and glucagon; together, these receptors form a distinct family called G-protein coupled receptors (GPCR/GPCRs) family B (Kolakowski L. F., Receptor Channels, 2, 1994, 1-7). The GPCR comprise an extracellular N-terminal domain of 100-160 residues, connected to a juxtamembrane domain (J-domain) of seven membrane-spanning α-helices with intervening loops and a C-terminal tail (Donnelly D., FEBS Letts., 409, 1997, 431-436). The Class B GPCRs are activated by endogenous peptide ligands of intermediate size, typically 30-40 amino acids (Hoare, S. R. J., Drug. Discovery Today, 10, 2005, 423-427). A general mechanism of peptide ligand interaction with class B GPCRs has emerged and is termed as the ‘two-domain’ model. The C-terminal portion of the peptide binds the N-domain of the receptor, confirm binding of ligand with the receptor and the N-terminal ligand region binds the J-domain, an interaction that activates the receptor and stimulates intracellular signaling (Ji T. H., et al., J. Biol. Chem.; 273, 1988, 17299-17302; Hjorth, S. A., et al., Regulatory Peptides, 64, 1996, 70).
PTH binds to the PTH receptor with affinity in the nM range; the ligand-occupied receptor transmits a signal across the cell membrane to intracellular effector enzymes through a mechanism that involves intermediary heterotrimeric GTP-binding proteins (G proteins). The primary intracellular effector enzyme activated by the PTH receptor in response to PTH peptide is adenylyl cyclase (AC) (Goltzman D., J. Bone Miner. Res., 15(3), 2000, 605-608). Thus PTH induces increase in the second messenger, cyclic adenosine monophosphate (cAMP) which regulates the poorly characterized downstream cellular processes involved in bone remolding (Juppner H., et al., Science, 254, 1991, 1024-1026). Other signalling pathways of this receptor, such as elevation of intracellular calcium, phospholipase C-dependent and independent activation of protein kinase C, have been described. Since PTH regulates blood calcium and the phosphate levels and exhibit potent anabolic (bone-forming) effects, the parathyroid hormone and its derivatives represent potential therapeutic agent for the treatment of osteoporosis (Slovik D. M., et al., J. Bone Miner. Res., 1, 1986, 377-381; Dempster D. W., et al., Endocr. Rev., 14, 1993, 690-709).
Synthetic PTH (1-34) exhibits full bioactivity in most cell-based assay systems, has potent anabolic effects on bone mass in animals and has recently been shown to reduce the risk of bone fracture in postmenopausal osteoporotic women. In human trials on postmenopausal women, daily subcutaneous injections of low doses of PTH (1-34) were shown to result in impressive bone formation in the spine and femoral neck with significant reduction in incidence of vertebral fractures (Neer R. M., et al., N. Engl. J. Med., 344, 2001, 1434-1441; Dempster D. W., et al., Endocr. Rev., 14, 1993, 690-709). These clinical data reveal PTH as one of the most efficacious agents tested for osteoporosis. Under the brand name Forteo (Eli Lilly), PTH (1-34) in the form of teriparatide acetate has been approved for the treatment of osteoporosis.
PTH derivatives include polypeptides that have amino acid substitutions or are truncated relative to the full-length molecule. Both the N and C-terminal truncated forms of PTH (1-34) has been studied. Additionally, amino acid substitutions within the truncated polypeptides have also been investigated. (Azurani A., et al., J. Biol. Chem., 271, 1996, 14931-14936). It has been known that residues in the 15-34 domain of PTH peptide contribute importantly to receptor binding affinity, while N-terminal 1-14 amino acids of PTH peptide are responsible for the activation of receptor (Naussbaum S. R., et al., J. Biol. Chem., 255, 1980, 10183-10187; Gardella T. J., et al., Endocrinology, 132, 1993, 2024-2030; Takasu H., et al., Biochemistry, 38, 1999, 13453-13460; Hoare S. R. J., et al., J. Biol. Chem., 276, 2001, 7741-7753; Luck M. D., et al., Molecular Endocrinology, 13, 1999, 670-680). Truncated PTH (1-34) derivatives such as cyclised PTH (1-17), PTH (1-28) and PTH (1-31) are active in most assay systems and promote bone-formation (Whitfield J. F., et al., J. Bone Miner. Res., 12, 1997, 1246-1252; WO 2007/130113 A2; WO 2008/068487; Whitfield J. F., et al., Calcif. Tissue Int., 56, 1995, 227-231; Rixon R. H., et al., J. Bone Miner. Res., 9, 1994, 1179-1189; Whitfield J. F., et al., Trends Pharmacol. Sci., 16, 1995, 372-386; Whitfield J. F., et al., Calcif. Tissue Int., 58, 1996, 81-87). But these peptides are still too large for efficient non-parenteral delivery. The discovery of an even smaller PTH agonist would be an important advance in the effort to develop new treatments for osteoporosis.
Unfortunately, due to the large molecular weight of PTH peptide, its therapeutic application has been limited, since its synthesis is technically difficult and therefore expensive and the only possible administration mode is the injection route. Moreover, PTH is highly susceptible to protease attack and must be stored at low temperature due to its low stability. In addition to these technical limitations, tolerability is limited by transient mobilization of calcium and hypercalcemia also the toxicological data and in particular the unfavourable results of cancerogenesis studies (dose and treatment duration dependent increased risk of osteosarcoma) induce a cautious use of PTH (1-34) (Vahle J. L., Toxicol. Pathos., 32(4), 2004, 426-438; Whitfield J. F., Medscape Womens Health, 6(5), 2001, 7; Kuijpers G., BMJ, 324(7335), 2002, 435-436). On the other hand, low molecular weight peptides, for instance those consisting of the first 14 or 11 amino acids of PTH (PTH(1-14) and PTH(1-11)), proved to be inactive or exhibited very low biological activity, in animal models (Tregear G. W., et al., Endocrinology, 93, 1973, 1349-1353; Gardella T. J., et al., J. Biol. Chem., 266, 1991, 13141-13146).
Therefore, during last decade, investigation has focused on development of PTH-derived low molecular weight peptides with improved biological profile, preferably orally bioavailable, protease resistant, easy to synthesis and exhibit a greater safety index. Recently, it was found that the activity of low molecular weight peptides can be improved by introducing helix stablising unnatural amino acids at specific positions. For example, PTH(1-11) analogs ([Ala3, Gln10, Arg11]-PTH(1-11), [Ala3, Gln10, Har11]-PTH(1-11) and [Aib1,3; Gln10; Har11]-PTH(1-11)) and PTH (1-14) analogs, such as [AC5C1, Aib3, Gln10, Har11, Ala12, Trp14]PTH(1-14) stimulate cAMP, in nM range (WO 03/009804; WO 04/093902). Several studies were carried out to find low molecular weight peptides with PTH-like activity (Reidhaar-olson J. F., et al., Mol. Cell. Endocrinology., 160, 2000, 135-147; Shimizu M., et al., J. Biol. Chem., 275, 2000, 21836-21843; Shimizu M., et al., Endocrinology, 142, 2001, 3068-3074; Shimizu N., et al., J. Biol. Chem., 276, 2001, 49003-49012; WO 03/009804). Although short analogues consisting of as little as 11 amino acids (derivatives of first 1-11 residues of PTH peptide, Seq. ID. No. 2) can activate the PTH receptor (in vitro) with low potency (WO 04/067021), however, in animal models (in vivo) bone-anabolic activity of these analogues has not been reported. In conclusion, agonist activity on cAMP-signalling pathway of the PTH receptor (in vitro) alone is not at all predictive for bone-anabolic activity in vivo.
In the present investigation, surprisingly, we found that homologous substitution (derivatives) of N-terminal sequence of PTH (1-34) peptide (first 1-14 or 1-15 residues, Seq. ID. No. 3 and 4) with unnatural amino acids resulted in the identification of novel class of short-chain peptides having potent PTH receptor agonistic activity, more specifically PTH-1 receptor agonistic activity, at varying degree of selectivity. To enhance the duration of action and stability against proteolytic enzyme, we have site-specifically modified the short-chain peptides with unnatural amino acids and succeeded in identifying metabolically stable and highly potent short-chain peptides. Some of the short-chain peptides showed bioavailability even by oral route of administration, while retaining PTH-1 receptor agonistic activities.
PTH (1-34) sequences alignment shown below represents the primary structural relationships:
PTH (1-34):(Seq. ID No: 1)1SVSEIQLMHNLGKH14LNSMERVEWLRKKLQDVHNF34. PTH (1-11):(Seq. ID No: 2)1SVSEIQLMHNL11 PTH (1-14):(Seq. ID No: 3)1SVSEIQLMHNLGKH14 PTH (1-15):(Seq. ID No: 4)1SVSEIQLMHNLGKHL15
Single-letter abbreviations for amino acids can be found in Zubay, G., Biochemistry 2nd ed., 1988, MacMillan Publishing, New York, p. 33.